Three years ago, a group of researchers began campaigning for the adoption of guidelines that promised to improve the reproducibility of RT-qPCR data. Lauren Arcuri Ware reports on the adoption and evolution of these guidelines.

Reverse-transcription quantitative PCR (RT-qPCR) has been the subject of
considerable controversy. While the technique is considered the gold
standard for quantifying gene expression in a cell, there are so many
variables involved that two researchers could perform the same experiment
and end up with wildly
different results. And although a study may produce a statistically
significant result, it's hard to know if that result is truly valid or if
the data might have been skewed due to a technical error.

A new set of MIQE guidelines geared to the specific concerns of digital PCR have recently been published in Clinical Chemistry. Source: Clinical Chemistry

It’s easy to perform PCR experiments, says Jim Huggett. "But the trouble is, there's an awful lot of upstream preparation needed and a whole multitude of things you can do beforehand which will alter the results you get." Source: Jim Huggett

In 2009, a group of researchers led by Stephen Bustin, a molecular biologist at Anglia Ruskin University, published guidelines in the journal Clinical Chemistry to help scientists publish data that are both accurate and reproducible. Source: Stephen Bustin

So, in 2009, a group of researchers led by Stephen Bustin, a molecular
biologist at Anglia Ruskin University, published guidelines in the journal Clinical
Chemistry to help scientists publish data that are both accurate and
reproducible (1). These guidelines are called the Minimum Information for
Publication of Quantitative Real-Time PCR Experiments, or MIQE for short,
and address several key aspects of qPCR, including sample quality control,
assay design, PCR efficiency, and normalization.

Of course, guidelines only work if they are widely adopted by labs and
journals and remain up-to-date with new technical developments. So where do
the MIQE guidelines stand now, three years after their initial publication?

Adoption Challenges

It’s easy to perform PCR experiments, says Jim Huggett, who leads the nucleic
acid metrology research group at UK-based biotechnology company LGC Limited. "But
the trouble is, there's an awful lot of upstream preparation needed and a
whole multitude of things you can do beforehand which will alter the results
you get." If researchers don't consider these factors—including
extraction method, sample storage conditions, and more—they may not realize
what is influencing the results.

Bustin knows all about results that are not what they appear to be. He
investigated studies that used qPCR to show the measles virus was present in
intestinal tissue of children with autism, implicating the measles, mumps,
and rubella (MMR) vaccine in the development of the disorder. Because the
researchers had skipped an important step in the protocols—they had
inadvertently neglected to add reverse transcriptase to several samples to
convert the measles virus RNA to DNA—Bustin was able to determine that the
samples were contaminated with DNA plasmids from a facility located next
door to the lab. The positive qPCR signal that the researchers had picked up
was from those plasmids, not the measles virus.

Although this well-publicized and extreme case demonstrates the potential
usefulness of the MIQE guidelines, many researchers still don't follow them. "We're
beginning to see people becoming more careful," says Bustin, "but
if you look at the numbers of papers that cite the MIQE guidelines, it's
about 7 percent in terms of qPCR, which means that 93 percent of papers are
still being published with inadequate information that doesn't allow anyone
to decide whether the data are real. And if you look at the data, you can
very often see that they are meaningless." For example, when a paper
reports a one-and-a-half fold difference in gene expression that is
normalized against the expression of a single unvalidated reference gene,
it's hard to conclude that the result is valid.

Despite the low percentage of papers citing the MIQE guidelines, one group is
taking the guidelines very seriously and advocating that researchers stick
to them: the companies that sell reagents and other qPCR supplies. "There's
clearly an improvement in the quality of the publications that are being
published [as a result]," says Huggett.

In a paper published recently in PLoS One, biomedical researcher
Francis Jacob from the University Hospital Basel in Switzerland and
colleagues reported their attempts to identify a set of suitable, reliable
reference genes for several different human cancer cell lines and to
determine whether MIQE guidelines are actually being followed by labs (2).
His group found that many of the studies look complete on the surface, but
when you delve into the details, you find that important data are missing.
Many papers don't report the efficiency of their reference genes or their
qPCR data, and only 30–40 percent of published studies that investigated
reference genes actually followed the MIQE guidelines.

New Digital Guidelines

Meanwhile, qPCR is ever evolving. Its newest incarnation, digital
PCR or dPCR, is being used by more and more labs. As a result, a new set
of MIQE guidelines geared to the specific concerns of this brand-new version
of PCR have recently been published in Clinical Chemistry (3).

Most researchers in the field concede that the original MIQE guidelines came a
bit too late in the process, after years of questionable qPCR data
reporting. With the advent of dPCR, Huggett says that the goal was to
publish guidelines early on in the adoption of this new version of the
technique.

Although it remains expensive and time-consuming, dPCR has gained some
traction with the development of microfluidics and nanofluidics as well as
with emulsion chemistries used in next-generation DNA sequencing. In
addition, dPCR is generally more precise than RT-qPCR, says Huggett. "It's
much more reproducible without the need for standard curves," he says. "It
has the potential to revolutionize quantification by using molecular methods."

But is it too early in the development of this new technique to publish
guidelines? The authors don’t believe so. They reasoned that dPCR does seem
to have legs as a technique and that certain dogmatic ideas about the
process are already becoming set in stone. "We are very much trying to,
at an early stage, get people thinking about the benefits of dPCR, while
still remembering there are some limitations, and most importantly, that you
need to prove to yourself that the technique is working well enough, rather
than just assuming it's fine," says Huggett.

Ultimately, the goal for the qPCR and dPCR MIQE guidelines is for the data to
become more transparent, says Bustin. When the data are more standardized,
other researchers can analyze them and come to their own conclusions about
the validity of the results. "My conversion to this was when I was
acting as an expert witness to the MMR trials," says Bustin. "I
had data. I had to report on it, and it wasn't clear how they had gotten
that data. Once I got access to the raw data and analyzed it myself, I was
able to understand how they had gone wrong."

The key, he emphasizes, is access to the raw data, and he believes that,
within five years' time, publishing raw data will be the standard procedure
for qPCR papers. "If you could import the [raw data] file into your
analysis program, then you could see right away whether you believe the data
or not. You can immediately come to conclusions about the quality of the
work. I think that's where we're headed."